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United States Patent |
5,149,647
|
Burling
|
September 22, 1992
|
Process for extracting pure fractions of lactoperoxidase and lactoferrin
from milk serum
Abstract
A process for extracting pure fractions of lactoperoxidase and lactoferrin
from milk serum is described. The milk serum is microfiltered and passed
through a strong cation exchanger at a high rate of flow for selective
adsorption of lactoperoxidase and lactoferrin, and then the
lactoperoxidase and lactoferrin are eluted successively and selectively
with saline solutions having different concentrations.
Inventors:
|
Burling; Hans (Lund, SE)
|
Assignee:
|
Svenska Mejeriernas Riksforenings Ekonomi-Aktiebolag (Stockholm, SE)
|
Appl. No.:
|
488040 |
Filed:
|
May 24, 1990 |
PCT Filed:
|
November 25, 1988
|
PCT NO:
|
PCT/SE88/00643
|
371 Date:
|
May 24, 1990
|
102(e) Date:
|
May 24, 1990
|
PCT PUB.NO.:
|
WO89/04608 |
PCT PUB. Date:
|
June 1, 1989 |
Foreign Application Priority Data
| Nov 27, 1987[SE] | 8704719-7 |
Current U.S. Class: |
435/192; 435/815; 530/366; 530/416 |
Intern'l Class: |
C12N 009/08; C07K 003/22; C07K 013/00; A23J 001/20 |
Field of Search: |
435/192,815
530/366,416
|
References Cited
U.S. Patent Documents
3896241 | Jul., 1975 | Malaspina et al. | 426/271.
|
4436658 | Mar., 1984 | Peyrouset et al. | 260/122.
|
4791193 | Dec., 1988 | Okonogi et al. | 530/416.
|
4946944 | Aug., 1990 | Frankinet et al. | 530/400.
|
Foreign Patent Documents |
2179947 | Mar., 1987 | GB3.
| |
Other References
Chemical abstracts, vol. 105, 1986, abstract No. 41350u, J Chromatogr.,
1986, 358(2), 429-433 (Eng).
Buzila et al, "The Simultaneous Preparation of the Active Components From
Human Milk," Rev. roum. Biochim., vol. 21, Iss. 2, pp. 81-91 (1984).
Boyer (1986) "Modern Experimental Biochemistry" pp. 49-53.
|
Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Weber; Jon P.
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier & Neustadt
Claims
I claim:
1. A process for extracting pure fractions of lactoperoxidase and
lactoferrin from milk serum, comprising initially microfiltering the milk
serum, then passing it through a bed of a fast flow type strong cation
exchanger at a high rate of flow of about 1-1.5 bed volumes/minute for
selective adsorption of lactoperoxidase and lactoferrin, and then
successively and selectively eluting the lactoperoxidase with a saline
solution having a concentration of 0.10-0.4 M at a pH of about 6.5 and the
lactoferrin with a saline solution having a concentration of 0.5-2 M.
2. The process as claimed in claim 1, where prior to the elution of
lactoperoxidase, the cation exchanger is eluted with a saline solution
having a concentration of 0.01-0.15 M.
3. The process as claimed in claim 1 or 2, wherein that the pH of the milk
serum is adjusted to 5.9-9.0, preferably about 6.5, before being passed
through the cation exchanger.
4. The process as claimed claim 1 wherein the microfiltration is carried
out in a microfilter having a pore diameter of 0.10-2 .mu.m.
5. The process as claimed claim 1 wherein the saline solutions with eluted
lactoperoxidase and lactoferrin, respectively, are concentrated, desalted
and freeze-dried.
6. The process as claimed in claim 2, wherein the microfiltration is
carried out in a microfilter having a pore diameter of 0.10-2 .mu.m.
7. The process as claimed in claim 3, wherein the microfiltration is
carried out in a microfilter having a pore diameter of 0.10-2 .mu.m.
8. The process as claimed in claim 1, wherein the microfiltration is
carried out in a microfilter having a pore diameter of 0.4-1.5 .mu.m.
9. The process as claimed in claim 2, wherein the microfiltration is
carried out in a microfilter having a pore diameter of 0.4-1.5 .mu.m.
10. The process as claimed in claim 3, wherein the microfiltration is
carried out in a microfilter having a bore diameter of 0.4-1.5 .mu.m.
11. The process as claimed in claim 2, wherein the saline solutions with
eluted lactoperoxidase and lactoferrin, respectively, are concentrated,
desalted and freeze-dried.
12. The process as claimed in claim 3, wherein the saline solutions with
eluted lactoperoxidase and lactoferrin, respectively, are concentrated,
desalted and freeze-dried.
13. The process as claimed in claim 4, wherein the saline solutions with
eluted lactoperoxidase and lactoferrin, respectively, are concentrated,
desalted and freeze-dried.
14. The process of claim 2 wherein said salt solution is a solution of an
inorganic alkali, alkaline earth or ammonium salt.
Description
The present invention relates to a process for extracting pure fractions of
lactoperoxidase and lactoferrin from milk serum. By milk serum is meant
both skim milk and whey.
In cheese-making, a large amount of whey is obtained as a by-product. Whey
has a dry solids content of about 6%, which is composed approximately as
follows:
______________________________________
% by weight
______________________________________
Lactose 4.6
Protein 0.6 thereof
Lactoperoxidase 0.0020
Lactoferrin 0.0030
Fat 0.05 (after separation)
Salts 0.7
Dry solids content about
6.0
______________________________________
The protein fraction which constitutes about 10-12% of the dry solids
content is composed of a number of different protein components The
biggest are .beta.-lactoglobulin, .alpha.-lactalbumin and bovine
serum-albumin. Also a number of bioactive components belong to the protein
fraction, for example immunoglobulins, lactoperoxidase, lactoferrin and
lysozyme.
Both lactoperoxidase and lactoferrin have antimicrobial properties. There
is a great interest in extracting natural antimicrobial substances to be
used in new contexts in food technology and in the chemico-technical and
medical fields.
There are low contents of these substances in skim milk and whey (also in
the original milk). Lactoperoxidase and lactoferrin are present in
contents of 15-50 mg/litre, depending on the lactation state of the cow.
Large quantities of whey (milk) must thus be filtered to facilitate
extration of kilogram amounts of these bioactive components.
The process engineering conditions for isolating lactoperoxidase and
lactoferrin, respectively, from milk/whey are based on the fact that the
isoelectric point (pI) for these two proteins is about 9.5, while the main
part of the whey proteins have isoelectric points around 5.1-5.4 and the
casein at about 4.6. A fundamentally suitable process for separation of
lactoperoxidase and lactoferrin is therefore to contact the milk/whey with
a cation exchanger at a pH of <6 for selective adsorption, and use is here
made of the positive net charge of lactoperoxidase and lactoferrin, which
distinguishes from that of other milk proteins which have a negative
charge at this pH.
The traditional way of isolating lactoperoxidase and lactoferrin in small
amounts for the purpose of research is to use the precipitation technique
and ion exchange chromatography, frequently combined with gel filtration,
see Morrison, M., Hamilton, H-B., Stotz, E., J. Biol. Chem. 228:767
(1957); and Morrison, M., Hultquist, P-E., J. Biol. Chem. 238-2847 (1963).
These methods are not suited for preparing large amounts of the bioactive
components at issue in an economically defensible manner.
U.S. Pat. No. 4,436,658 (Pevrosuset) discloses adsorption of lactoferrin
from casein-free milk serum (whey) by means of a silica column. The pH of
the milk serum is adjusted to 7.7-8.2 before adsorption on the column.
Immunoglobulins, lactoferrin and lactoperoxidase adhere to the column.
After the adsorption phase, elution with a diluted saline solution at a pH
of <4 takes place. No selective elution of the adsorbed proteins is
obtained, particularly not regarding lactoperoxidase. A column holding
about 5 g of silica compound can treat 1 litre of whey. This prior art
process must be regarded as unsuitable for application on an industrial
scale.
Zagulski et al. in Prace in Materialy Zootechniczne 20, (1979), p. 87-103
describes a batchwise method of obtaining lactoferrin, in which use is
made of a weak cation exchanger which is mixed with milk. After
equilibration, the ion exchanger is applied to a column for elution of the
adsorbed proteins with a saline solution. The method thus is based on a
batchwise process, and a further purification must be carried out in a
second ion exchange step to obtain a high purity of the lactoferrin.
A similar process is described in BE patent specification 901,672 (J. P.
Prieels and R. Peipper, Oleofina S.A.). Here, use is made of an ion
exchanger based on calcium alginate, in which the ion exchange
functionality has been obtained by admixture of oxides of zirconium,
titanium, quartz or aluminium. The milk/ whey is contacted with the ion
exchanger in a packed column or by mixing in a tank, whereby proteins
having an isoelectric point above 7.5 are adsorbed. After equilibration,
the gel is separated mechanically and supplied to a means for washing and
eluting with a calcium chloride solution. All fluids contacting the
calcium alginate gel must contain at least 0.1% CaCl.sub.2 to prevent the
gel from being dissolved. No fractionating of lactoperoxidase and
lactoferrin is obtained in the elution, but the fractionating must be
carried out in a separate purification step.
As a reason for not working with a commercially established ion exchange
technique in a column process, the above-mentioned BE patent specification
mentions the unsurmountable difficulties of clogging of the ion exchanger
caused by the occurrence of particles of globular fat and protein
aggregate in the medium.
GB patent specification 2,179,947 discloses a process for the extraction of
lactotransferrin from milk. The process is carried out such that the whey
is subjected to ultrafiltration, whereby the protein content of the whey
(including the lactotransferrin) is concentrated about 5 times, whereupon
its pH and ionic strength are adjusted. The milk serum thus treated is
passed at a very low rate (about 0.03 bed volumes per minute) through an
ion exchange column, preferably a weak cation exchanger. The column is
eluted, still at a low rate, with a solution having an ionic strength
gradient which increases up to 0.4 M, when the lactotransferrin is eluted.
This is a small-scale process which is not suitable for industrial
preparation of lactoferrin. The use of a weak cation exchanger results in
a poor capacity. Whey in an amount of 100 bed volumes, converted into a
natural dry solids content, can pass through the ion exchange column
between each elution. The problem of clogging of the ion exchange filter
caused by fat and protein particles has not been solved by this prior art
process.
The following requirements can be placed on an industrially applicable
process for economic recovery of lactoperoxidase and lactoferrin from
whey/skim milk:
1) High selective capacity of the adsorption mass. Since the contents of
lactoperoxidase/lactoferrin are low in milk serum, the volumes of milk
serum which can be treated in one elution, must be large.
2) High rate of flow in the adsorption phase. (Normal chromatographic
processes usually work at low rates, 0.01-0.10 bed volumes per minute. The
reason is that owing to the small particle size, the bed usually gives
high pressure drops, and that the reaction kinetics for the adsorption
process frequently require a high rate of flow.
3) The process must be hygienic, which means that the adsorption mass must
stand at least a lye treatment at pH 13-14.
The object of the present invention is to achieve a process that satisfies
the above-mentioned requirements for extraction of pure fractions of
lactoperoxidase and lactoferrin from milk serum (whey) on a large scale
and at a low cost.
The present invention relates to a process for extracting pure fractions of
lactoperoxidase and lactoferrin from milk serum, said process being
characterised by microfiltering the milk serum, passing it through a
strong cation exchanger at a high rate of flow for selective adsorption of
lactoperoxidase and lactoferrin, and successively eluting the
lactoperoxidase and the lactoferrin selectively with saline solutions
having different concentrations.
According to the invention, a process is provided for preparing pure
fractions of two different serum proteins in a single ion exchange step.
This has not previously been achieved on an industrial scale. Prior art
methods for extracting these proteins on an industrial scale have required
two or three purification steps.
The above-mentioned problems of clogging of the ion exchanger, which is
caused by the occurrence of particles, such as globular fat and protein
aggregates, in the serum or whey, are solved according the invention in
that the milk serum (whey) is microfiltered, for example in a so-called
cross-flow process, before contacting the ion exchange bed. By choosing a
suitable pore size of the microfilter, fat and protein aggregate particles
which cause clogging, can be eliminated. A suitable microfilter has a pore
diameter of 0.10-2 .mu.m, preferably 0.4-1.5 .mu.m.
As the starting material for the process according to the invention, milk
serum (whey) is used, i.e. milk freed from fat and casein. The milk serum
is first treated by microfiltration for removal of residues of fat and
protein aggregate particles, preferably in a so-called cross-flow process.
The microfiltered milk serum is then passed at a high rate (about 1-1.5
bed volumes per minute) through a column packed with a strong cation
exchanger which selectively adsorbs lactoperoxidase and lactoferrin. This
cation exchanger has excellent rate and adsorption kinetic properties and
a capacity of about 1000 bed volumes of milk serum. This means that about
1000 bed volumes of milk serum can pass before the lactoperoxidase which
has the weakest bond, breaks through, i.e. the ion exchange mass is
saturated with these proteins. Merely a slight increase of the pressure
drop occurs between the beginning and the end of the adsorption phase.
The elution of the ion exchange mass is started by washing the milk serum
out of the column with a buffer, preferably a phosphate buffer at the pH
of the milk serum, 6.5. Subsequently, impurities, if any, are eluted with
a buffer solution containing a weak saline solution, preferably of an
inorganic alkali, alkaline earth or ammonium salt, for example 0.075 M
NaCl.
After this preparatory elution, the desired proteins are selectively eluted
with buffer solutions containing saline solutions selected from the
above-mentioned salts, at different concentrations. Thus, the elution of
lactoperoxidase is performed at a salt concentration in the range of
0.10-0.4 M, and of lactoferrin at a salt concentration within 0.5-2 M.
After this treatment, the proteins concerned have been concentrated about
500 times.
The pure protein fractions are collected, and then a further concentration
is preferably effected by ultrafiltration followed by desalination and
freeze-drying so as to obtain a commercial product consisting of about 90%
pure protein fractions.
For the production of 1 kg lactoperoxidase and 1 kg lactoferrin, about 65
and, respectively, 45 m.sup.3 of whey are required. The purity of the
extracted components exceeds 90%. This is obtained by a suitable choice of
ion exchanger and a careful choice of adsorption and elution conditions in
which the pH and the salt concentrations are important parameters.
The invention will now be described in detail by means of the Example below
and the accompanying drawings.
FIG. 1 is a schematic view of a preferred embodiment of the process
according to the invention;
FIG. 2 illustrates the UV absorption spectrum when eluting lactoperoxidase
and lactoferrin from an ion exchange column; and
FIGS. 3a and 3b are chromatograms showing the purity of lactoperoxidase and
lactoferrin after the fractionating according to the invention has been
carried out.
EXAMPLE
100 litres of pasteurised and sludge-centrifuged sweet whey at pH 6.5 were
microfiltered in a cross-flow process at 50.degree. C. By the
microfiltration, remaining residues of globular fat were removed together
with occurring protein aggregates. The pore size of the microfilter was
1.4 .mu.m.
After cooling, the whey was passed through an ion exchange column packed
with 80 ml of a specially treated strong cation exchanger (S-Sepharose,
fast flow, Pharmacia) on an agarose basis. The height of the bed was about
4.1 cm, and the rate through the column was 100 ml/minute, corresponding
to a rate of 1.25 bed volumes per minute. The pressure drop before the
column at the beginning of the run was 0.26 bar. 15 h later, the rate was
still 100 ml/minute at a pressure drop of 0.28 bar. The lactoperoxidase
break-through occurred when about 80-90 litres of whey had been passed
through the column, i.e. about 1000 bed volumes.
Subsequently, the flow of whey was interrupted, and the eluting phase was
started by washing the whey out of the column with a phosphate buffer,
0.01 M KH.sub.2 PO.sub.4, pH 6.5, followed by elution of impurities from
the ion exchanger with a phosphate buffer containing 0.075 M NaCl (FIG.
1). The lactoperoxidase was eluted with a phosphate buffer containing 0.3
M NaCl, and then the lactoferrin was eluted with a phosphate buffer
containing 0.9 M NaCl (see FIG. 2).
After the fractions had been collected, they were desalted by gel
filtration in a Sephadex column and were finally freeze-dried.
The ion exchange column was cleaned by washing first with 2.0 M NaCl and
then with 1.0 M NaOH, whereupon the column was ready for the next run.
______________________________________
Yield of lactoperoxidase after ion exchange:
96.5%.
Purity of the collected fraction after elution:
A.sub.412/A280 = 0.84*
Total yield in the process after
90%
freeze-drying, calculated as activity:
Purity of the freeze-dried
A.sub.412/A280 = 0.87*
preparation:
______________________________________
*0.92 is the maximum quota for 100% purity.
The corresponding yield and purity were obtained for the lactoferrin (see
FIGS. 3a and 3b).
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